Purification of blood coagulation factors
The present invention relates to the purification of vitamin K-dependent blood coagulation factors, such as Factor IX (FIX). In particular, the invention provides a method for purifying Factor IX having a desired content of gamma-carboxyglutamic acid from a sample comprising a mixture of species of said Factor IX having different contents of gamma-carboxyglutamic acid, said method comprising the steps of: (a) loading said Factor IX sample onto an immunoaffinity chromatography material coupled to a binding moiety for gamma-carboxyglutamic acid; (b) eluting said Factor IX; and (c) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids; characterized in that the total concentration of Factor IX within said sample exceeds the binding ability of the immunoaffinity chromatography material.
1. A method for purifying a sample of human Factor IX, comprising:
(a) loading a sample comprising different species of human Factor IX onto an immunoaffinity chromatography material comprising a gamma-carboxyglutamic-directed antibody (Gla-directed antibody);
wherein the amino acid sequence of the Gla-directed antibody comprises SEQ ID NO: 1 and SEQ ID NO: 2;
wherein the different species of human Factor IX comprise different numbers of Gla residues;
(b) eluting the sample of human Factor IX; and
(c) selecting a fraction obtained from the elution comprising an increase in the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX compared with the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX in the sample being purified;
wherein the total concentration of human Factor IX within the sample exceeds the binding ability of the immunoaffinity chromatography material.
2. The method according to claim 1 , wherein the binding ability of the immunoaffinity chromatography material is exceeded by a percentage selected from the group consisting of 100, 105, 110, 115, 120, 125, 130, 140, 150, 200, 250, 500, 750, and 1000%.
3. The method according to claim 1 , wherein the binding ability of the immunoaffinity chromatography material is exceeded by between 100-400%.
4. The method according to claim 1 , wherein the fraction comprises a decrease in the proportion of #1-10-Gla species of human Factor IX compared with the proportion of #1-10-Gla species of Factor IX in the sample being purified.
5. The method according to claim 1 , wherein the eluting comprises loading an elution buffer at a pH of between 5.0 and 8.5.
6. The method according to claim 1 , wherein the eluting comprises an elution buffer comprising sodium chloride at a concentration of between 10 mM and 100 mM.
7. The method according to claim 1 , wherein the divalent cation is calcium or magnesium.
8. The method according to claim 1 , wherein the immunoaffinity material comprises a sepharose bead.
9. The method according to claim 1 , further comprising loading an equilibration buffer prior to loading the sample and loading a wash buffer after loading the sample, wherein the equilibrium buffer and the wash buffer each comprises a calcium ion.
10. The method according to claim 9 , wherein the calcium ion is present in a concentration greater than 0.5 mM.
11. The method according to claim 1 , further comprising loading an equilibrium buffer prior to loading the sample at a pH of between 5.0 and 8.5.
12. The method according to claim 9 , wherein the calcium ion is calcium chloride.
13. The method according to claim 1 , further comprising selecting a fraction obtained from the eluted sample of Factor IX as a second sample and further purifying the second sample using anion chromatography.
14. A method for purifying a sample of human Factor IX, comprising:
loading a sample comprising different species of human Factor IX onto an immunoaffinity chromatography material comprising a gamma-carboxyglutamic-directed antibody (Gla-directed antibody);
wherein the amino acid sequence of the Gla-directed antibody comprises SEQ ID NO: 1 and SEQ ID NO: 2;
wherein the total concentration of human Factor IX within the sample exceeds the binding ability of the immunoaffinity chromatography material;
b) eluting the sample of human Factor IX;
c) selecting a fraction obtained from the elution as a second sample of human Factor IX;
d) loading the second sample of human Factor IX onto an anion exchange chromatography material;
e) eluting the second sample of human Factor IX; and
f) selecting a fraction obtained from the elution of the second sample comprising an increase in the proportion of one or both high Gla species #1-11-Gla and #1-12-Gla of human Factor IX compared with the proportion of one or both of #1-11-Gla and #1-12-Gla species of human Factor IX in the sample being purified.
15. The method of claim 13 , wherein eluting the second sample is done using an eluent selected from the group consisting of ammonium acetate, ammonium chloride, sodium acetate, and sodium chloride.
16. The method of claim 13 , wherein eluent is at a pH between about 5.0 to about 8.5.
17. The method according to claim 13 , wherein the second sample of human Factor IX comprises a decrease in the proportion of #1-10-Gla species of human Factor IX compared with the proportion of #1-10-Gla species of Factor IX in the sample being purified.
18. The method according to claim 13 , wherein the divalent cation is calcium or magnesium.
19. The method according to claim 13 , wherein the immunoaffinity material comprises a sepharose bead.